The syndrome of resistance to thyroid hormone (RTH) is caused by multiple distinct mutations in the ligand-binding domain of the thyroid hormone receptor-beta (TRbeta). Although the mutant receptors are transcriptionally inactive, they inhibit normal receptor function in a dominant negative manner to cause hormone resistance. Recently, a group of transcriptional cofactors, referred to as corepressors (CoRs), was shown to induce ligand-independent silencing of genes that contain positive T3 response elements. CoRs also play a role in the ligand-independent basal activation of genes that are negatively regulated in response to T3. We hypothesized that CoR might play a role in the dominant negative inhibition by TRbeta mutants that cause RTH. In gel mobility shift assays, RTH mutants retained interactions with CoRs even in the presence of T3, whereas the ligand dissociated CoR from wild-type TRbeta. Using Gal4-TR chimeric receptors and a VP16-CoR fusion protein in an interaction assay, a strong positive correlation was found between mutant receptor interactions with CoR and transcriptional silencing activity. A mutation (P214R) that impairs CoR interactions with TR was introduced into the RTH mutants to assess the role of CoR in dominant negative activity. In transient transfection assays, introduction of the P214R CoR mutation decreased RTH mutant silencing of positively regulated genes and basal activation of negatively regulated genes. The dominant negative activity of several different RTH mutants, studied by cotransfection with wild-type receptor, was greatly diminished by the CoR mutation, and this effect was seen with both positively and negatively regulated genes. These results suggest that CoR interactions play a critical role in the dominant negative effect of RTH mutants and support the idea that these proteins are involved in the regulation of genes that are positively as well as negatively regulated by T3.